Post-transplantation lymphoproliferative disorders (PTLD) are life-threatening complications of solid organ transplantation (SOTx), caused by Epstein Barr Virus (EBV) infections and the use of chronic non-specific immunosuppression (IS). The incidence of PTLD in patients who are EBV seronegative prior to transplantation, mostly pediatric patients, is as high as 25%. The onset of the disease is usually preceded by an elevated EBV load in the peripheral blood which is highly sensitive but not specific for the diagnosis of PTLD. Our group and others have shown that routine long-term post-Tx viral load monitoring identifies a group of children who carry persistent very high viral loads for months to years after primary post-Tx EBV infection. We recently showed that these chronic high EBV load asymptomatic carriers have a 45% rate of progression to late-onset PTLD, including diffuse large B cell, Hodgkin's and Burkitt's lymphomas. To date, there is no clear understanding of how these viral loads are occurring or maintained, nor how we can predict which patients will develop chronic high viral loads and progress towards PTLD/lymphoma. Here we propose to elucidate the immunologic mechanisms responsible for this phenomenon and define novel prognostic immunologic signatures that can be used in the clinic to identify patients at risk of progression towards PTLD. In the first Aim we will assess the phenotypic and functional features of exhausted EBV-specific CD8+ and CD4+ T cells in pediatric transplant patients carrying chronic high EBV load, as compared to low or absent EBV load carriers. In Aim 2 we will characterize the co-regulation of exhausted EBV-specific CD8+ T cells by inhibitory receptors and by helper ? chain family cytokines in chronic high EBV load pediatric transplant patients, as compared to low or absent EBV load carriers. In Aim 3 we will investigate intrinsic molecular mechanisms underlying exhaustion of EBV- specific CD8+ T cells in pediatric transplant patients carrying chronic high EBV load, and compare results to those from low or absent EBV load carriers. These studies will provide a rational basis for future implementation of cellular monitoring in the clinic to identify exhausted EBV-specific CD8+ T cells and the risk of progression to PTLD. These studies could also provide support for early therapeutic intervention (lowering IS) and for the development of novel therapeutic approaches to reverse CD8+ T cell exhaustion and improve outcomes in pediatric Tx patients. PUBLIC HEALTH RELEVANCE: This research will improve the understanding of immunologic and molecular mechanisms causing for certain high chronic EBV load pediatric HTx patients to display impaired EBV-specific immunity, and to be at higher risk to progress towards PTLD. Our goal is not only to identify useful immunologic biomarkers that might help monitor and predict which patients are at risk of progression towards malignancy, but also to reveal the molecular pathways that may be targeted with novel therapeutic agents to prevent or improve the outcomes of PTLD in the pediatric transplant setting.